The present invention is directed to labeled oligonucleotides, methods for making the same, and compounds useful therefor. More specifically, this invention relates to oligonucleotides selectively functionalized at one or more of the 3xe2x80x2-terminalnucleotide, 5xe2x80x2-terminalnucleotide, and internucleotides with conjugate groups, methods for making the same, and compounds useful therefor.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. The widespread use of such oligonucleotides has increased the demand for rapid, inexpensive and efficient procedures for their modification and synthesis. Early synthetic approaches to oligonucleotide synthesis included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72, 209, 1972; Reese, Tetrahedron Lett. 34, 3143-3179, 1978. These approaches eventually gave way to more efficient modern methods, such as the use of phosphoramidites and H-phosphonates. Beaucage and Caruthers, Tetrahedron Lett., 22, 1859-1862, 1981; Agrawal and Zamecnik, U.S. Pat. No. 5,149,798, issued 1992.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most popular processes is the phosphoramidite technique (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
The phosphoramidite technique, however, has significant disadvantages. For example, cyanoethyl phosphoramidite monomers are quite expensive. Although considerable quantities of monomer go unreacted in a typical phosphoramidite coupling, unreacted monomer can be recovered, if at all, only with great difficulty.
The ability of the acylaminoethyl group to serve as a protecting group for certain phosphate diesters was first observed by Ziodrou and Schmir. Zioudrou et al., J. Amer. Chem. Soc., 85, 3258, 1963. A version of this method was extended to the solid phase synthesis of oligonucleotide dimers, and oligomers with oxaphospholidine nucleoside building blocks as substitutes for conventional phosphoramidites. Iyer et al., Tetrahedron Lett., 39, 2491-2494, 1998; PCT International Publication WO/9639413, published Dec. 12, 1996. Similar methods using N-trifluoroacetyl-aminoalkanols as phosphate protecting groups has also been reported by Wilk et al., J. Org. Chem., 62, 6712-6713, 1997. This deprotection is governed by a mechanism that involves removal of N-trifluoroacetyl group followed by cyclization of aminoalkyl phosphotriesters to azacyclanes, which is accompanied by the release of the phosphodiester group.
Solid phase techniques continue to play a large role in oligonucleotidic synthetic approaches. Typically, the 3xe2x80x2-most nucleoside is anchored to a solid support which is functionalized with hydroxyl or amino residues. The additional nucleosides are subsequently added in a step-wise fashion to form the desired linkages between the 3xe2x80x2-functional group of the incoming nucleoside, and the 5xe2x80x2-hydroxyl group of the support bound nucleoside. Implicit to this step-wise assembly is the judicious choice of suitable phosphorus protecting groups. Such protecting groups serve to shield phosphorus moieties of the nucleoside base portion of the growing oligomer until such time that it is cleaved from the solid support. Consequently, new protecting groups, which are versatile in oligonucleotidic synthesis, are needed.
A variety of modifications to naturally occurring oligonucleotides have been proposed. Such modifications include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents. Other mechanisms of action have also been proposed.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
For example, antisense oligonucleotides have been modified to be conjugated with lipophilic molecules. The presence of the lipophilic conjugate has been shown to improve cellular permeation of the oligonucleotide and, accordingly, improve distribution of the oligonucleotide in cells. Further, oligonucleotides conjugated with lipophilic molecules are able to enhance the free uptake of the oligonucleotides without the need for any transfection agents in cell culture studies. Conjugated oligonucleotides are also able to improve the protein binding of oligonucleotides containing phosphodiester linkages.
Recently, oligonucleotides selectively labeled with two different reporter groups have attained a widespread interest due to their unique properties. For example, if the reporter groups are paired as a donor and an acceptor of fluorescent energy, a fluorescence resonance energy transfer (FRET) between the two groups may occur. When the distance between the donor and acceptor groups is short, quenching of the fluorescence is observed. In contrast, when the donor and acceptor are sufficiently far apart, a fluorescent signal is observed. Such a phenomenon has been used to detect formation of a complex between a suitably labeled oligonucleotide and a complementary target nucleic acid. When the oligonucleotide is uncomplexed, the donor and the acceptor groups are sufficiently close so that no fluorescence is detected. However, when the oligonucleotide complexes with the nucleic acid, the donor and acceptor groups are forced to move away from each other, thereby restoring the fluorescence signal. Oligonucleotides that exhibit such a hybridization-dependent fluorescence have been termed xe2x80x9cmolecular beacons.xe2x80x9d Molecular beacons may be useful in numerous applications, such as real-time monitoring of hybridization in PCR, in molecular biosensors, on surfaces, in blood, in living cells, and in vivo. Synthesis of double labeled oligonucleotides has been performed with the aid of dye-labeled solid supports and phosphoramidites. In addition, oligonucleotides that bear an amino and an activated thiol group at opposite ends have been synthesized using modified phosphoramidites and solid supports, where chemoselective labeling is performed post-synthetically. However, the known methods for synthesizing double labeled oligonucleotides are restricted to the use of only certain dyes that are available as phosphoramidite, solid support, and chemoselective reagents.
In light of the foregoing, there is a continued need for selectively labeled oligonucleotides, methods for making the oligonucleotides, and compounds useful therefor. The labeled oligonucleotides should provide improved cellular permeation, enhanced free uptake of the oligonucleotide in cell culture studies, and improved protein binding, especially for oligonucleotides containing phosphodiester linkages. In addition, there is a need for methods for producing oligonucleotides selectively labeled at one or more of the 3xe2x80x2-terminal nucleotide, 5xe2x80x2-terminal nucleotide, and internucleotides with one or more different conjugate groups. The methods should also provide for such labeled oligonucleotides without the need for post-synthetic labeling.
The present invention allows for the selective functionalization of oligonucleotides with conjugate groups. In particular, the oligonucleotides can be selectively functionalized with a first conjugate group at the 3xe2x80x2-terminal nucleotide and optionally functionalized with a second conjugate group at the 5xe2x80x2-terminal nucleotide and/or one or more internucleotides. Alternatively, the oligonucleotides can be selectively functionalized with a first conjugate group at the 5xe2x80x2-terminal nucleotide and optionally functionalized with a second conjugate group at one or more internucleotides. In yet another embodiment, the oligonucleotides can be functionalized with a first conjugate group at one or more internucleotides and with a second conjugate group at one or more different internucleotides.
It is an object of the present invention to provide oligonucleotides having the formula: 
wherein:
R1 is hydroxyl, a protected hydroxyl or a group having the formula: 
Q0 is O or S;
R4 Oxe2x88x92, hydroxyl or a protected hydroxyl;
R2 is hydroxyl, a protected hydroxyl or a group having the formula: 
each R3 is H, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group;
each X is, independently, Oxe2x88x92, hydroxyl, protected hydroxyl or xe2x80x94Sxe2x80x94L3;
each Bx is an optionally protected heterocyclic base moiety;
n is from 3 to about 50; and
L1, L2 and each of said L3 are, independently, a conjugate group
It is a further object of the present invention to provide methods for the preparation of selectively functionalized oligonucleotides having the above formula.
It is yet a further object of the present invention to provide synthetic intermediates useful in such methods.
Other objects will be apparent to those skilled in the art.